The vanadium effect in nitrogen fixation by azotobacter

25

BIOCHIMICA ET BIOPItYSICA ACTA

BBA 26816 T H E VANADIUM E F F E C T IN N I T R O G E N F I X A T I O N BY AZOTOBACTER j . R. B E N E M A N N ,

C. E. M c K E N N A ,

R. F. L I E , T. G. T R A Y L O R

AND M. D. K A M E N

Department of Chemistry, University of California, San Diego, La Jolla, Calif. 92 037 (U.S.A.) (Received O c t o b e r i 8 t h , 1971)

SUMMARY

In some strains of Azotobacter vanadium will substitute for the Mo requirement of N2-fixing cell cultures. This V effect was studied using two strains of Azotobacter: A. vinelandii OP and A . chroococcum ATCC 480. Growth rate determinations showed that V could be about 5o % as effective as Mo in stimulating growth on N 2. However, determination of nitrogenase activity in actively growing cultures indicated that the V effect produces only about IO % of the activity of Mo-grown cells. The specific activity, and stability toward storage, air, and heat, of the nitrogenase in cell-free extracts of V-grown Azotobacter cultures were intermediate between those of Mogrown and Mo-starved cell-free extracts. Purification of V-nitrogenase revealed V to be incorporated into the nitrogenase enzyme as part of a Mo-Fe like type protein which contained V and Mo. On the basis of the activity to Mo ratios in extracts from Mo-limited cultures we conclude that the Mo contained in V-nitrogenase substantially accounts for its observed activity. We propose that the V effect is due to V incorporation into the nitrogenase ccmplex with consequent stabilization of the enzyme and more effective utilization of the small amount of Mo found in Mo-starved cells.

INTRODUCTION

The molybdenum (Mo) requirement of N2-fixing bacteria, first discovered by Bortels 1 for Azotobacter, is due to the presence of this metal in one of the two proteins of nitrogenase ~. Bortels a also found that vanadium (V) could partially satisfy the Mo requirement of some Azotobacter species. This "V effect" has never been explained on a molecular basis and is the subject of this paper. Mo is required by all microorganisms capable of utilizing N 2 (refs I, 4-11) and has been found as a constituent of all nitrogenases so far studied2,12-14. V, however, can substitute for Mo only in the case of some strains of several species of aerobic nitrogen-fixhag organisms: Azotobacter vinelandii, Azotobacter chroococcure 3, ln-17 and Mycobacterium flavum TM. It cannot substitute for the Mo requirement in N z fixation by other Azotobacter species 15, Beijerinckia species15,19, 2°, or bluegreen algae21; unconfirmed reports exist that it can support N 2 fixation in other bacteria4, s. The V effect appears to be specific for N 2 fixation; growth on nitrate, which also requires Mo, is reportedly not supported by V in those strains able to use it for N~ fixation 15. Recently, McKenna et al. 2~ showed that cell-free extracts 13iochim. Biophys. Acta, 264 (1972) 25-38

26

J.R. BENEMANN et al.

of A . vinelandii OP grown on N2 in the presence of V contained, after dialysis, both V and Mo (in a ratio of about 93 to 7) and that the nitrogenase in these extracts ("V-nitrogenase") exhibited lower acetylene binding affinity than nitrogenase from Mo-grown cells ("Mo-nitrogenase"). Burns et al3, using the same organism, confirmed the difference in Michaelis-Menten constants (Kin) for acetylene between N o - a n d V-grown nitrogenase preparations and also reported that V-nitrogenase, when compared to Mo-nitrogenase, exhibited higher ratios of H 2 evolution to substrate reduction, differing ratios of products of acrylonitrile reduction, slightly lower activitation energies, and less sensitivity to CO inhibition. Both groups concluded from their data that V was incorporated into nitrogenase where it affected both specific activity and substrate binding and that this implicated Mo in the substrate binding and reduction reactions of nitrogenase. The question of whether V was actually effective in the nitrogenase catalysis was not settled. McKenna et al. 22 found ratios of nitrogenase activity to Mo content three to five times higher in V-nitrogenase than in Mo-nitrogenase. Although this could indicate that V contributes to the catalytic activity of V-nitrogenase, other possible explanations, such as loss of Mo during dialysis of V-nitrogenase preparations or No bound to proteins other than nitrogenase in Mo-nitrogenase extracts, could also account for this observation. Burns et al3 ~, while presenting only qualitative data for the metal content of their preparations, claimed that only lO% of the activity of V-nitrogenase preparations could be accounted for by Mo contamination. Attempts to purify V-nitrogenase into its constitutive proteins resulted in loss of both activity and metal content ~4. Consequently the nature of the V effect remained unexplained. In this paper we have studied the properties of nitrogenase from No-starved and V-grown A . vinelandii OP. We have also succeeded in separating the proteins of V-nitrogenase from A. chroococcum ATCC 48o and found that the N o - F e like protein contained V in a ratio of V to Mo of 4 to I. Our results lead to an explanation for the V effect. MATERIALS AND METHODS

The following Azotobacter species were tested for presence of a V effect: A . vinelandii OP (ATCC137o5) ; A . vinelandii 0 (obtainecl from Prof. R. C. Valentine) ; A . chroococcum ATCC 9544; A . chroococcum ATCC 480, ICPB 3157 (obtained from Prof. M. Starr); A . agilis (from Prof. R. C. Valentine) and A. chroococcum C44

(Indiana State Culture Collection, provided by Mr. G. Miura). The Azotobacter strains were grown on a modified Burk's medium containing in deionized water I mM MgSO4, 4 mM KH~PO 4 (adjusted to p H 7-4 with KOH), o.2 mM CaC12 and o.o 7 mM Fee13. The carbon source was ethanol (2 %) in the case of A. vinelandii OP and sucrose (2 %) for A. chroococcum ATCC 48o. Molar stock solutions of the minerals were purified by the methods of Arnon et al. 2~ and Delwiche et al36 to reduce Mo contamination. The glassware used in cell culture was soaked with 2 M KOH, rinsed with deionized water, soaked with 6 M HC1, and finally rinsed several times with deionized water. The purified medium was designated "nilmedium"; "V-medium" contained 25 ° ppb V (V20 ~ dissolved in dil. H2SO4); "3Iomedium" contained 25o ppb No (Na2MoO4). Growth rate studies were performed by inoculating Azotobacter cultures Biochim. Biophys. Acta, 264 (i972) 25 38

THE VANADIUMEFFECT IN AZOTOBACTER

27

(5 °o inoculum) into 25 ml of medium in 125 ml Erlenmeyer flasks with a sidearm and incubating at 3 °0 on a New Brunswick G-33 Gyrotary Shaker at 3 cycles/sec. Growth was followed by measurement of turbidity using a Klett-Summerson colorimeter with a 66o-nm filter. For preparation of cell extracts the Azotobacter cultures were transferred (lO-2O % inoculum at each step) into successively larger volumes of media with final inoculation into 2o-1 glass carboys. A. chroococcum ATCC 3157 was grown, prior to the transfer into carboys, with I ppb. Mo. Aeration was vigorous with cells growing on Mo, sparing with cells grown on nil-media or V-media. The cultures were harvested by means of a Sharples ultracentrifuge when the Klett reading reached at least 80. The cell paste was suspended in a 5-fold volume of ice-cold 0.025 M ph¢:sphate buffer (pH 7.5) and centrifuged, and the washed pellet resuspended in a 2-fold volume of the buffer and broken in a French press. The crude extract was centrifuged (I h, 30000 × g) to remove whole cells and debris before assay or further purification. Metal analyses were performed on extract or protein samples which had been freeze-dried, refluxed with 3 ml conc. H~SO 4 in Kjeldahl flasks for 8-12 h, decolorized with HNO3, and finally diluted to a known volume with water. Preliminary assays for the metals were done by atomic absorption followed by colorimetric determinations. Mo and V were determined by modifications 27 of the methods of Johnson and Arkley ~ and Talvitie 29, respectively. Mo analysis of the media indicated that although the minerals contributed, at most, 0.04 ppb Mo, the final Mo contamination in the ethanol nil-media was about 0.2 ppb. Sucrose contributed about 0. 5 ppb to the media and the V salt contained 0.025 % Mo. To assay nitrogenase activity in whole cells I ml of the cell culture was injected into the assay bottle, containing a 20 % 02-80 % Ar gas mixture, followed by i ml of acetylene which started the reaction. Cell-free extracts were assayed by first injecting i ml acetylene, followed by the extract and water to give a total reaction volume of i ml, then 20 /,moles of Na2S20 * (neutralized), and finally the ATP generator (containing per assay 5 #moles ATP, 0.2 mg creatine phosphokinase, 5 pmoles MgC12, 25 /,moles creatine phosphate and 50 /,moles H E P E S buffer, p H 7.4) to initiate the reaction. Reactions were terminated by injection of 0.25 ml of 25 % trichloroacetic acid. Ethylene was determined by injecting a gas sample from the assay bottle into a Hewlett-Packard FM7oo gas chromatograph containing a 6 ft × 1/8 inch Poropak-N column and equipped with a flame ionization detector and a Sargent SR6 recorder with disk integrator. Specific activities were calculated, using acetylene as an internal standard, with help of a computer program 27. Protein concentrations were determined by the Biuret method 3°. Nitrogenase was purified by a modification e7 of the method of Bulen and LeComte 2. RESULTS The V effect in Azotobacter cultures

Of several strains, of Azotobacter tested (see Materials and Methods) only A. vinelandii OP and A. chroococcum ATCC 480 exhibited V-stimulated growth on purified nil-medium (Fig. I). The V effect was most pronounced with A. chroococcum ATCC 480 since this strain exhibited no growth on nil-medium. A. vinelandii OP Biochim. Biophys. Acta, 264 (1972) 25-38

28

J . R . BENEMANN et al.

grew slowly on nil-medium because the Mo c o n t a m i n a t i o n was a b o u t o.2 ppb, while the h a l f - m a x i m a l g r o w t h r e q u i r e m e n t d e t e r m i n e d for this strain was only o.8 ppb. The concentrations of V required to s t i m u l a t e g r o w t h of N2-fixing A z o t o b a c t e r cultures are s o m e w h a t higher t h a n those of Mo b u t on the same order of m a g n i t u d e : I to IO p p b V were effective. 200

I

I

I

I

I

I

b

a

0

I00

0

Itl I

80

lo

/,/" /~

60

2 / •, , -

40

/

/

,Io

20

Io o

I0

0

I0

20

30

0

TIME

I I0

I 20

I 30

40

(HOURS)

Fig. I. Effect of added Mo or V on growth of Azotobacter cultures. Growth was followed after three mediuIn-consistent transfers. (a) A. chroococcum ATCC 48o. (b) A . vinelandii OP. © - - : ~ , purified medium + 25° ppb Mo; B - - B , purified medium -F 25° ppb V; 0 - - 0 , purified medium + o.o 5 ppb Mo; ~--KI, purified medium alone (nil-medium). M a x i m a l g r o w t h r a t e s t i m u l a t i o n b y V was always below t h a t of Mo (Fig. I). T o t a l g r o w t h was also lower on V- t h a n on Mo-medium. The level of Mo c o n t a m i n a t i n g the V salt (0.02 %) h a d no effect on growth. Therefore the V effect was not due to this factor, in a g r e e m e n t with previous findings 15. Whole cell acetylene r e d u c t i o n activities were s t i m u l a t e d b y V even less t h a n g r o w t h r a t e s when c o m p a r e d to Mo controls (Table I). These d a t a i n d i c a t e d t h a t g r o w t h rates were not d i r e c t l y c o m p a rable to nitrogenase a c t i v i t y i n vivo. S u b s t a n t i a l g r o w t h rates could be achieved with c o m p a r a t i v e l y low rates of nitrogenase activity. Whole cell assays of Mo-starved cultures e x h i b i t e d poor reproducibility, even a m o n g duplicates, a n d r a n g e d between 0 and 3 % of t h e a c t i v i t y of cells growing on Mo-media. The reason for this is t h a t the nitrogenase a c t i v i t y of Mo-starved cells is v e r y labile u n d e r the assay conditions. I n c u b a t i o n in an A r - O ~ - a c e t y l e n e a t m o s p h e r e stops further N2 fixation a n d therefore protein synthesis, resulting in a r a p i d decline of nitrogenase a c t i v i t y due to 02 Biochim. Biophys. Acta, 264 (1972) 25-38

29

THE VANADIUM EFFECT IN AZOTOBACTER

inactivation. Studies of the regulation of Azotobacter nitrogenase have demonstrated nitrogenase activity not to be the limiting factor in the growth of nitrogen-fixing cultures (when sufficient Mo was present) and that early exponential cultures were fixing more N 2 than required for cell growth a. It is apparent therefore that the V effect, when measured directly by N 2 fixation of cell cultures, is much smaller than previously estimated for experiments comparing total growth or static cultures 3, la, 17 'FABLE I GROWTH

RATES

A.ND

NITROGENASE

ACTIVITIES

OF A.

vinelandii O P

CULTURES

G r o w t h r a t e s a n d specific activities d e t e r m i n e d after 4 t r a n s f e r s in different e x p e r i m e n t s . Media

D o u b l i n g t i m e (h) o.,o

Nil

V

Mo

9.8 32

6.0 55

3.3 ioo

Specific a c t i v i t y (nmoles e t h y l e n e / m l per rain)

o.15

o/ ,o

0. 5

3.i IO

32 100

T A B L E II ~Io CONTENT OF AZOTOBACTER CELLS Values r e p r e s e n t n m o l e s 3Io/mg p r o t e i n in crude e x t r a c t s . Strain

d . chroococcum ATCC 480 ,4. vinelandii O P

Media Nil

V

o.oi26 0.0325

o.oi69 0.0548

Azotobacter cells grown on V-medium contain about 50 % more Mo than when grown on nil-medium (Table II). Although significant, this result by itself cannot account for the much higher increases in growth rates and nitrogenase activities observed (both in whole cell and cell-free extracts). Addition nitrate to Mo medium completely repressed nitrogenase activity; however, its addition to V medium results in cultures which exhibit whole cell nitrogenase (acetylene reduction) activities equal to those observed in the absence of nitrate. Since nitrate repression of nitrogenase biosynthesis is effected,.by the N H 3 produced from it, this indicated that V did not significantly stimulate nitrate reduction, otherwise a repression of nitrogenase activity would have been observed. If V-stimulated uptake of Mo into Azotobacter cells occurs, nitrate utilization should be stimulated by the presence of V, especially since nitrate reduction has a lower Mo requirement than N 2 fixation15' 32,38. Therefore, the V effect cannot be due to an increased Mo cell uptake and, as reported before 15, is specific for N 2 fixation. Biochim. Biophys. ,4cta, 264 (i972) 25-38

30

J . R . BENEMANN el al.

The i n h i b i t o r y effect of W (Na2W04) on Mo m e t a b o l i s m is well knowna4, a~ a n d has been s t u d i e d in N 2 fixation b y A z o t o b a c t e r ~ - a . T u n g s t a t e affects Moa n d V-grown nitrogen-fixing A z o t o b a c t e r cultures differently. T u n g s t a t e inhibition is c o m p e t i t i v e in the presence of Mo (Ki 1.7. I°-7-2-°" 1°-7 M for A . vinelandii OP) a n d Mo can overcome W inhibition. However, V cannot overcome W inhibition at a n y c o n c e n t r a t i o n (Table III). Similar results r e p o r t e d previouslya< a7 were not conclusive because an A z o t o b a c t e r s t r a i n was used which did not exhibit a V effect ag. The t u n g s t a t e e x p e r i m e n t s i n d i c a t e d a significant difference in the mode of action of V a n d Mo which was not a p p a r e n t from simple g r o w t h experiments. TABLE

III

INHIBITION BY \ V OF GROXVTH B Y A . v i n e l a n d i i

OP

ON LOV¢-.~IO-, V- AND NIL-MEDIUM

Values shown are averages for duplicate flasks.

Purified medium phts :

Nil Mo, Mo, V, V, Mo,

2.0 200 2.0 200 2.0

Growth (Klett) z 4 h after transfer

*

3:0 W

ppb ppb** ppb ppb*** ppb and V, 200 ppb

[WI z.8.zo ~ 3I

41 87 ~oS 46 0~ So

3 3 86 2 2 2

* Contained "2% sucrose. **

=

2.1

" IO -6

.\~.

= 3.9" 10-6 ~'[' TABLE

IV

NITROGENASE ACTIVITIES OF AZOTOBACTER EXTRACTS S p e c i f i c a c t i v i t i e s ( n n l o l e s e t h y l e n e / m i n p e r m g p r o t e i n ) of c e n t r i f u g e d e x t r a c t s , P r o t e i n a m o u n t s u s e d in t h e a s s a y s i n d i c a t e d i n p a r e n t h e s e s .

Strain

A. chroococcum ATCC 480 A. vinelandiiOP

3[edia Nil

V

340

I.o (I2.8) o. 5 ( 8 . 2 )

5.4 (5 .°) 5.8(II.3)

26.4 (6.4) 28.9 (5.9)

V-nitrogenase: activity and lability in crude extracts

Cell-free e x t r a c t s of A z o t o b a c t e r grown on V - m e d i u m h a d lower nitrogenase activities t h a n those grown on Mo-medium, b u t much higher activities t h a n those of n i l - m e d i u m grown cells (Table IV). The specific activities of the cell-free e x t r a c t s of V-nitrogenase a n d nil-nitrogenase e x h i b i t e d considerable v a r i a t i o n from experim e n t to e x p e r i m e n t . C o m p a r e d to Mo-nitrogenase t h e y ranged between 0.5 a n d 4 % for nil-nitrogenase a n d b e t w e e n 5 a n d 20 % for V-nitrogenase. The r a t i o s of activities of Mo-nitrogenase a n d V-nitrogenase in crude e x t r a c t s correspond a p p r o x i m a t e l y to t h e a c t i v i t y r a t i o s of whole cell assays. Biochim. Biophys. Mcta, 264 (1972) 25-38

T H E V A N A D I U M E F F E C T IN A Z O T O B A C T E R

31

The rates of loss of activity for V-nitrogenase and nil-nitrogenase preparations exposed to air and Ar are shown in Table V. Exposure to air resulted in the loss of most or all V-nitrogenase and nil-nitrogenase activity while Mo-nitrogenase (not shown in the table) lost no activity after 21 h. Even under Ar the loss of Vnitrogenase and nil-nitrogenase activity was substantial. In both cases, and with both strains of Azotobacter, V-nitrogenase activity was more stable than nil-nitrogenase. The heat lability of the various nitrogenases (Table VI) followed the same pattern. Because of the low activities in the crude extracts of V-nitrogenase and, TABLE

V

LOSS OF AZOTOB&CTER NITROGENASE ACTIVITY UNDER ARGON AND AIR Specific activities are relative to values in Table IV.

Strain

Metal added to medium

% Loss of activity 5 h

IIh

Ar

Air

Ar

21 h Air

Ar

Air

A. chrooeoceum A T C C 4 8 0

V None

3° 25

80 97

35 28

92 ioo

60 48

99 IOO

,4. vinelandii O P

V None

18 3°

29 83

24 42

42 69

3° 83

87 ioo

TABLE

VI

HEAT STABILITY OF AZOTOBACTER NITROGENASE Specific activities are relative to those given in Table IV.

Strain

Medium

% Loss of activity at 60 ° zo rain

20 rain

A. vinelandii O P

Mo V None

o 25 69

io 80 i oo

A. chroococcum A T C C 4 8 0

Mo V None

o 5° 94

o 75 ioo

especially, nil-nitrogenase, the data in Tables V and VI must be considered to be qualitative. However the pattern which the data show is unmistakable and reproducible, and agrees with the observations with whole cells: presence of V in Modeficient growth medium results in nitrogenase activity which is substantially higher and more stable than that observed in its absence. The lability of nil-nitrogenase and V-nitrogenase cannot arise from the effect of some factor present in these extracts because mixing them with extracts from Mo-grown cells does not affect the activity of Mo-nitrogenase. Diluting Mo-nitrogenase to activities observed with nil-nitrogenase makes Mo-nitrogenase unstable, suggesting that a mere dilution effect could Biochim. Biophys. Acta, 2 6 4 (1972) 2 5 - 3 8

32

j.R. BENEMANNgl al.

account for the observations reported in Tables V and VI. However, these dilution effects are primarily due to protein dilution; as long as the protein concentration is high, Mo-nitrogenase is relatively stable even in low concentrations. Since nilnitrogenase and V-nitrogenase activities were measured in crude (centrifuged) cellfree extracts, dilution by itself cannot account for the data. Similarly, the high concentration of oxidizable proteins (such as the Fe-protein of nitrogenase) in the crude extracts would exclude the possibility that small amounts of contaminating 0 2 result in a larger percentage of nitrogenase inactivation in the low activity extracts than in the high activity extracts, thereby accounting for the observed results. F e - p r o t e i n i n A zotobacter cells g r o w n on V - a n d n i l - m e d i u m

The effect of the addition of purified Fe-Mo protein of Azotobacter v i n e l a n d i i OP to crude extracts of the two Azotobacter strains grown on all three media is shown in Table VII. This experiment is in fact an assay for the Fe protein of nitrogenase because Fe-Mo is active only in the presence of the Fe protein. The results indicate that the Fe protein is present in extracts from Mo-starved Azotobacter in amounts (considering protein concentration) somewhat over half of those found in Mo-grown extracts. Thus, V-nitrogenase and nil-nitrogenase are not deficient in Fe-protein, but rather appear to lack an active Fe-Mo protein. TABLE VII E F F E C T OF

Mo-Fe

PROTEIN

DEAE

F R A C T I O N ON A C T I V I T I E S ( A C E T Y L E N E ) O F C E L L - F R E E

EXTRACTS

FROM .4. vinelandii OP AND A. chroococcum ATCC48o GROWNON Mo-. V- OR NIL-MEDIU~.I Activities are given relative to those in "Fable IV. % Activity

Cell-free extract Strain

A. chroococcum ATCC 480

3¢edium

:\;o 3lo-Fe protein added

~,~lo-Feprotein (0. 5 rag) added

~'Io

1o o

12 9

V Nil A. vinelandii OP

~[O

V Nil

20.5 3.S I OO

20. 4 1.7

7I 12I II7

74 74

Evidence that the Fe-protein was unchanged in V-nitrogenase was also obtained from similar experiments with chromatographed V-nitrogenase of A. chroococcum ATCC 48o (see below). Indeed, the Fe-protein isolated during this purification was completely inactive by itself. As Fe-protein isolated from Mo-nitrogenase is contaminated by the Fe-Mo protein in sufficient amounts to give substantial activities, V-nitrogenase can be a good source of active, uncontaminated Fe-protein. The fact that Azotobacter continues to synthesize the Fe-protein of nitrogenase under conditions of Mo starvation indicates that the low activities observed with V- and nil-nitrogenase are not due to the inhibition of nitrogenase biosynthesis. Further support comes from the recent observation of W. Brill (personal communiBiochim. Biophys. Acta, 264 (1972) :25-38

33

THE VANADIUM EFFECT 1 N A z o T O B A C T E R

cation) that antigen which cross-reacts with the Fe-Mo protein antibody is found in normal amounts in cell extracts from Mo-starved Azotobacter cultures. Effect of Mo and V on A. vinelandii OP Mo content and nitrogenase activity The Mo content and specific activities of cell-free extracts of A. vinelandii OP grown on various levels of Mo with and without V is summarized in Table V I I I . As seen previously (Table III), the nitrogenase activity in crude extracts from V-grown cells is more than Io-fold higher than in the nil-controls, while Mo content differs by only 60 %. This again supports the view that V-stimulated Mo uptake does not account for the V effect. Most of the Mo present in the nil-nitrogenase extract is not tightly bound to protein; passage of the extracts (after centrifugation) through Sephadex G-25 results in an 80 % loss of Mo. Although the analytical data are relatively inaccurate (indicated by brackets) at these low metal and activity concentrations, the ratio o f activity to bound Mo is close to that of V-nitrogenase. In the V-nitrogenase extract Mo is not lost upon treatment with Sephadex G-25, indicating that it is bound much more strongly. TABLE VIII NITROGENASE ACTIVITIES AND ~O CONTENTS OF CELL-FREE EXTRACTS OF A. v i n e l a n d i i O P GRO~A'N ON VARYING CONCENTRATIONS OF ]~[O AND \~ V added to m e d i u m (ppb) : M o added to m e d i u m (ppb) : Specific activity:

o o o.2

C r u d e extracts nmoles Mo/mg protein in wh ole cells Activity/Mo

0.027 8

o 2. 5 4.2

0.077 54

C e n t r i f u g e d a n d S e p h a d e x G-25 treated extracts nmoles Mo/mg protein (o.oo5) 0.08o Activity/Mo (4 ° ) 54

o Ioo 32

500 o 3.5

500 I.O 3.9

500 ioo 35

1.o 4 31

0.044 80

0.067 58

1.98 18

3.1 io

0.056 62

0.077 .50

3.8 9.2

Addition of small concentrations of Mo to the nil-medium increases Mo content and specific nitrogenase activities and results in tighter binding of Mo to protein. A similar (but smaller) addition to V-grown cells increases Mo concentration in the extract somewhat but otherwise has little effect. High concentration of Mo in the media (above IO ppb) increases the Mo content of cells drastically. Mo is apparently stored in the cells bound to proteins other than nitrogenase, and the ratio of specific activity to Mo content drops sharply. The ratios of activities to bound Mo, shown in Table viii, indicate that, within experimental error, all the activity of V-nitrogenase can be accounted for by the Mo contamination. There is no need to postulate V catalysis to account for the V effect. Moreover, the cause of the V effect is also clear from the data in Table V I I I : V increases the ability of A. vinelandii OP to utilize and bind the available Mo in an active nitrogenase. In its absence most of the Mo present is not active in nitrogenase and not tightly bound to protein. Previously published experimental results22, 23 which showed high ratios of V-nitrogenase activity to Mo content must B i o c h i m . B i o p h y s . A c t a , 264 (1972) 25-38

34

j.R.

BENEMANN gt al.

take into account the obviously high level of Mo uptake in cells growing on Ioo pph Mo. The almost 2-fold difference in Mo content in cells grown on Ioo ppb Mo with and without V is not very significant as Mo content of cells depends on the culture conditions and cell densities at time of harvest.

Purification of V-nitrogenase Purification of V-nitrogenase has presented serious difficulties because of the low activity and unstable nature of the enzyme in the crude extracts, a fact also noted by Burns and H a r d y ~4 whose preparations lost all metal content and activity upon purification of V-nitrogenase from A. vinelandii OP. A. chroococcum ATCC 48o V-nitrogenase proved somewhat more amenable to purification; the results are shown in Table IX. The most important findings are that during purification both V and Mo are concentrated and present in the analogue of the Fe-Mo protein (designated "Fe--V-Mo protein"). TABLE IX METAL CONTENT AND NITROGENASE ACTIVITY OF PURIFIED V-NITROGENASE FROM A, ehYoococcitiyl ATCC 48o

Preparation

Centrifuged e x t r a c t After protanaine sulfate fractionation (PSII) After D E A E c h r o m a t o g r a p h y (Fe-V-IV[o protein)

V

,rio

nmoles/mg % protein (V + Mo)

nmoles/mg % protein (V + Mo)

o,88

98

O.Ol 7

2.3

83

o.48

17

28.1

4.°

81

o.94

19

(i )

2.o

Specific activity

1.89

Mo content in the P S I I fraction (nitrogenase precipitated with protamine sulfate and resolubilized by cellulose phosphate) was 28-fold higher than that present in crude extract. This impressive purification of Mo was accompanied by a I5-fold increase in specific activity, which, considering the large losses of activity (about 50 %) during this step in the purification, could have accounted for all the nitrogenase activity recovered. The specific V content also increased, though not as much as Mo content. It is likely that V was absorbed and stored in cells in large amounts bound to protein other than nitrogenase; therefore the specific V concentration in the first step of the purification would not have increased much. Indeed, the V content and purification in these experiments paralleled those of Mo in nitrogenase from cells grown on Mo-containing media. Chromatography of the P S I I material increased both Mo and V concentrations in the final product (the Fe-V-Mo protein) about 2-fold. This parallel purification of the metals during chromatography provides strong evidence that both are present in nitrogenase. The ratio of one mole of metal per 200 ooo g of protein in V-nitrogenase agrees with the metal content of Mo-nitrogenase in a comparable purification. This strongly suggests that V is indeed substituting for Mo in nitrogenase. The reasons for the great loss of activity during the chromatography are not known; it could arise from manipulation or from an inherent instability of the enzyme. However, this Biochim. Biophys. Acta, 264 (I972) 25-38

THE VANADIUM EFFECT IN AZOTOBACTER

35

does not detract from the central finding that nitrogenase from V-grown cells contains both V and Mo. Furthermore, the great degree of purification and high content of Mo in the active P S I I fraction of V-nitrogenase supports our view that V-nitrogenase activity is due to its Mo content. DISCUSSION AND CONCLUSIONS

Previous studies of the V effect in nitrogen fixation by Azotobacter have defined only the effect of the metal on total growth of the cultures3,Ll~-l~. In this paper we show that V also stimulates the growth rate of cells growing on Mo-deficient medium. Although not as large as when total growth is considered, the V effect on growth rates is quite substantial resulting in growth rates about half of those observed in the presence of Mo. However, direct assay of the nitrogenase activity b y Azotobacter cells indicates that the V effect is not as strong as previously thought ; whole cells of Azotobacter growing on V media have only about IO % the activity of the Mo controls. Nevertheless, this is still greater that that seen in the absence of any added metal, especially for A. chroococcum ATCC 480 which fails to grow to any significant extent on purified media. We have approached the problem of the V effect by comparing nitrogenase activity and Mo content in cell-free extracts of cultures growing under severe Mo starvation conditions to those of similar cultures growing with added V. The most notable effect of Mo starvation was a very low specific nitrogenase activity in the extracts, coupled with an extreme lability of this nil-nitrogenase activity on storage, or on exposure to heat or 0 2. Azotobacter nitrogenase, from cells grown with sufficient Mo, is an unusually stable enzyme. It is probable that the organization of the nitrogenase proteins into a large complex which can be sedimented by centrifugation (z5oooo × g for 6 h) is the reason for its stability. Indeed, when Azotobacter nitrogenase is purified, the complex is soluhilized and loses its heat, storage and O~ resistance properties *. Although most of the activity of nil-nitrogenase is lost upon ultracentrifugation (15000o × g for 6 h), that remaining is found only in the supernatant. It appears, therefore, as though the nil-nitrogenase activity behaves more like the purified enzyme than does the native nitrogenase complex from Mo-grown cells. Addition of V to the nil-medium results in a nitrogenase preparation (V-nitrogenase) which has about Io times higher activity and is substantially more stable. V-nitrogenase is intermediate in properties between nil-nitrogenase and Mo-nitrogenase in activity and stability. Centrifugation of V-nitrogenase usually results in activities being found in both supernatant fraction and pellet. Thus the V effect, under conditions of Mo starvation, increases and stabilizes nitrogenase activity. Nil-nitrogenase appears to utilize Mo in the cell inefficiently. Thus the ratio of activity to Mo present in the cell is low and most of this Mo can be removed by such gentle treatment as passage through Sephadex G-25. Addition of V to the medium results in a io-fold increase in the ratio of specific activity to Mo content owing to a large increase in specific activity. Also the Mo in the V-nitrogenase extracts is not lost upon treatment with Sephadex G-25. Thus V promotes a more effective utilization of the available Mo. We believe this to be the basis for the observed V effect. When Mo is added in low concentrations (2.5 ppb) to nil-media, one observes results similar to those seen with V; thus, specific activity increases drastically, Biochim. Biophys. Acta, 264 (i972) 2 5 - 3 8

36

J.R. BENEMANN el a[.

much faster than the Mo content, and the Mo present is not lost by Sephadex treatment. Besides increasing nitrogenase activity and stability and promoting Mo utilization, V also increases Mo uptake into the cell. Although this effect is small, amounting only to about a 50 % increase, it undoubtedly contributes to the overall V effect. The addition of small amounts of Mo to nil-media results in a large (2o-fold)increase in nitrogenase activity while Mo content increases only about 3-fold. It is clear therefore that modest increases in Mo content produce large changes in nitrogenase activity. However, a 2- to 3-fold increase in Mo content of nil-media grown cells would be required to account for the V effect. We do not believe, therefore, that V-promoted uptake of Mo into tile cell is primarily responsible for the V effect. Relevant evidence comes from two additional observations: V does not promote nitrate utilization and V is incorporated into nitrogenase. The ratio of nitrogenase activity to bound Mo (that remaining after passing the extract through Sephadex G-25) is, considering unavoidable variations in experimental parameters, remarkably constant in A . vinelandii OP cell-free extracts from cells grown on low Mo- or nil-media with and without V. The V effect is not necessarily ascribable to actual catalytic activity of V in nitrogenase, because all the activity can be accounted for by the presence of Mo. Although V-nitrogenase extracts had the highest ratios, they were not much higher than those seen in extracts from cells grown on low Mo, lending further support to the theory that V promotes more effective Mo utilization rather than filling the role of a catalyst. Further evidence against V catalysis in nitrogenase comes from the purification of A . chroococcum V-nitrogenase, wherein all the Mo on purification appears in the V-Mo-Fe protein, thereby proving that the 3Io observed in the crude extracts is actually part of nitrogenase. It is of course possible that V possesses small catalytic activity which is masked by the presence of Mo, but which is negligible with reference to the V effect. The presence of V in V-nitrogenase must therefore have significance other than as an actual catalyst. The stabilization of the nitrogenase activity suggests that the presence of V can serve a structural role in the nitrogenase complex. Although little is known about the organization, composition or rationale of the Azotobaeter nitrogenase complex, it clearly represents an adaptation of a strictly anaerobic enzyme system to the aerobic environment in which Azotobacter grows and which endows Azotobacter nitrogenase with its unusual properties. Tile biosynthesis of this complex, but not of the nitrogenase proteins, appears to be inhibited by Mo starvation and, at least partially, restored in the presence of V. V also affects some of the catalytic properties of Azotobacter nitrogenase, such as substrate binding 2~-,23, electron allocations, acrylonitrile reduction and CO inhibition 2~. The presence of V in the nitrogenase complex can account for these observations, if one invokes conformational changes. However, some of these observed differences between V-nitrogenase and Mo-nitrogenase might arise from effects of Mo starvation rather than of V replacement. Because nil-nitrogenase has low specific activity and is not stable, it is difficult to compare its properties to V-nitrogenase or Mo-nitrogenase. A comparison of the catalytic properties of purified and "native" Azotobacter nitrogenase might answer these questions. Although V is found in nitrogenase, it does not have a marked affinity for tile 13iochim. Biophys..4cta, 264 (i972) 25-38

THE VANADIUM EFFECT IN AZOTOBACTER

37

enzyme, compared to Mo. This is evident from the lack of a mass action effect. Thus even traces of Mo are incorporated into nitrogenase in the presence of high concentrations of V (indeed, this fact helps to account for the V effect). The \\; inhibition of the V effect also can be accounted for by our findings. If V is not catalytically active, then any interaction between the two metals need not affect N ~ fixation (as measured b y growth). The physiology and ecology of the V effect remains to be elucidated. The reason that only Azotobacter exhibits a V effect can be ascribed to the unique nature of its nitrogenase. Why some strains of Azotobacter exhibit a V effect while others do not is not known. The possibility that some strains are unable to incorporate V into nitrogenase should be probed experimentally. The importance of the V effect in nature also requires evaluation. We hazard the suggestion that, as V is found usually in much higher abundance than ~Io, the V effect might have a distinct advantage in localities where No is limiting. ACKN OWLEDGMENTS

This investigation was aided by National Institutes of Health grants I Fo2-GM37, 784 -oI (to J. R. Benemann); U.S.P.H.S. 2-Toi-oio45 (to C.E. McKenna) ; HD-oI262 (to M. D. Kamen); AM-II4o 4 (to T. G. Traylor); and National Science Foundation grant GA-25o5 o (to M. D. Kamen). We wish to thank Joel Goodman and Paul Kostel for technical assistance with some of the experiments described. REFERENCES 2 3 4 5 6 7 8 9 to 1t 12 ~3 14 15 i6 17 18 19 2o 2t 22 23 24 25

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